Study on the construction of pre-vascularized cell membrane sheets based on human osteoblastic cells
Objective:To explore a new method for constructing vascular network in vitro to solve the problem of pre-vascularization of engineered bone tissue.Methods:Frozen jaw human bone osteoblasts(HOBs)and human umbilical vein endothelial cells(HUVECs)were incubated with 0,5,25 and 50 μg/mL Fe3O4 MNPs.CCK-8 kit was used to detect the via-bility of HOBs derived from the cryopreserved bone of jaw and HUVECs under treatment of different concentrations of Fe3O4 MNPs.Prussian blue staining was used to measure the endocytosis of Fe3O4 MNPs.HOBs and HUVECs were incubated with 50 μg/mL Fe3O4 MNPs,and cell survival was detected with live/dead cell double staining kit at day 0,1,and 3.After incu-bation with 50 μg/mL Fe3O4 MNPs for different time(0,30 min,l h,and 2 h),HOBs and HUVECs were attracted by N41 supermagnet which was placed in the bottom of the dish,the adherent sheet formation of HOBs and HUVECs was detected by phalloidin staining.HOBs and HUVECs were cultured with 50 μg/mL Fe3O4 MNPs for 1 h,the sheet formation was controlled by magnetic attraction,and divided into HOBs+HOBs control group and HOBs+HUVECs+HOBs pre-vascularization group.3,3-dioctadecyloxacarbocyanine perchlorate(DiO)and 1,1'-Dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate(DiL)was used to lable HOBs and HUVECs for tracking.On day 3 and 7,Western blot and immunofluorescence staining method were performed to detect protein expressions and positive distribution area of bone morphogenetic protein-2(BMP2)and vascular endothelial growth factor-A(VEGF-A)in prevascularized cell sheets.Results:Compared with the cell control group,there was no significant difference in the growth of HOBs and HUVECs treated with 5,25,and 50 μg/mL Fe3O4 MNPs.Compared with cell control group,the endocytosis of nanoparticles were obviously observed in 50 μg/mL Fe3O4 MNPs group.Compared with the control group,there was no significant difference in the mortality of HOBs and HUVECs after 50 μg/mL Fe3O4 MNPs treatment.Compared with the unlabeled control group and the incubation group of 30 min,HOBs and HUVECs incubated at 50 μg/mL Fe3O4 MNPs for 1 h and 2 h showed more cell adherent sheet formation.Compared with the HOBs+HOBs control group,HOBs+HUVECs+HOBs pre-vascularization group showed obvious sandwich-like layered struc-ture and significantly increased membrane thickness,accompanying by increased expression levels and positive staining of VEGF-A and BMP2 proteins.Conclusion:Prevascularized cell sheets of the osteoblast was formed in vitro by nanoparticle la-beling and magnetic attraction,which provides a new theoretical guidance for optimizing the construction of prevascularized bone tissue.
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